Systems and Methods of Identifying Biomarkers for Subsequent Screening and Monitoring of Diseases

ABSTRACT

A system for generating an image of ultrastructural biomarkers from a biological sample is provided. The system includes a grid onto which a sample to be imaged may be placed and a cryogenic reservoir into which the grid and sample may be immersed for vitrification of the sample. The system also includes a stage onto which the grid and sample may be situated for subsequent imaging in a high contrast imager to permit identification of ultrastructural biomarkers therein. A method for generating an image of ultrastructural biomarkers from a biological sample is also provided. The generated image of ultrastructural biomarkers may be used subsequently for screening and monitoring diseases, evaluating drug and therapeutic efficacy, and assessing risks associated with a drug or therapeutic candidate, among other things.

RELATED U.S. APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationSer. Nos. 60/612,713 filed Sep. 24, 2004, which application is herebyincorporated herein by reference.

TECHNICAL FIELD

This invention relates generally to imaging systems and methods, andmore particularly to imaging systems and methods for identifyingultrastructural biomarkers for subsequent screening and monitoring ofdiseases.

BACKGROUND ART

The deepening productivity crisis in the pharmaceutical industry, thehigh cost to the pharmaceutical industry of introducing new drugs to themarket, partly because of expenses related to Phase I, II and IIIclinical trials, as well as late stage failures for many drug candidateshave spurred intense across-the-board activity around biomarkerdiscovery and validation. Biomarkers, defined by the FDA as acharacteristic that is objectively measured and evaluated as anindicator of normal biologic or pathogenic processes or pharmacologicalresponses to a therapeutic intervention, are being sought actively tohelp make early and cost-effective “go/no-go” decisions on drugs, forpatient stratification, clinical trial analysis, and finding nichemarkets (e.g., sub-population of patients who respond to drugs or inwhom no drug-related toxicity is seen) for new drugs under development.In addition, the FDA has recently recommended that validated orinvestigational biomarker data be included in IND and NDA packages.These are powerful drivers for the biomarker market, whose size isestimated at $428 millions in 2005, and is growing at 20 percent peryear.

The use of biomarkers is rapidly gaining momentum in the pharmaceuticalindustry and in the medical management of patients. Current methods foridentifying biomarkers involve the use of biochemical assays foridentifying “functional” biomarkers, such as genes or protein arrays ormetabolite analysis. The use of biochemical assays in this contextrequires probing for functional alterations in genes and proteins, theneed for a priori knowledge of their function, as well as extensiveassay development and optimization.

While there has been an explosion of biomarker discovery effortsutilizing genomics, proteomics and metabolomics, these technologies alsofocus only on functional biomarkers. With many diseases, the presence ofobservable functional biomarkers often occurs late in the disease state.As such, preventive measures for these diseases may be ineffective whendeveloped in connection with the management of the disease, or in earlyevaluation of drug efficacy.

Contributions towards understanding ultrastructural morphology have beenmade in recent years. Such an approach focuses on the ultra-structuraldifferences in the biological samples that can occur much earlier in thediseased state, even before functional differences are observable. Sincethese target structures typically range from between about 5 nanometers(nm) and 1 micrometer, one approach to visualize them is through the useof conventional transmission electron microscopy (TEM). However, the useof conventional TEM has some critical limitations. For example (i) thehigh vacuum used in TEM removes solvent, leaving behind structures thatare quite different from those present in the original solution, (ii)adequate contrast between the sample features and background is usuallynot available, necessitating the use of stains (the addition of stains,which usually are heavy metal salts, can cause dramatic changes inaggregate morphology), and (iii) the exposure of the sample to theelectron beam often damages the sample.

Accordingly, it would be desirable to provide an approach that cangenerate substantially artifact-free images of structural biomarkers ofa cell or biological sample without compromising the integrity of thebiomarkers in the sample.

SUMMARY OF THE INVENTION

The present invention provides, in one embodiment, an approach throughthe use of cryogenic transmission electron microscopy (cryo-TEM), aswell as modified freeze fracture direct imaging (M-FFDI) to identifyultra-structural biomarkers, which may subsequently be used forscreening and monitoring a range of diseases. The use of cryo-TEM andM-FFDI can generate substantially artifact-free images, unlike imagesobtained from conventional TEM.

In accordance with one embodiment of the present invention, a system forgenerating an image of ultrastructural biomarkers from a biologicalsample is provided. The system includes a grid onto which a sample to beimaged may be placed. The grid may be perforated so that a thin film ofthe sample may be generated across a hole. The system also includes acryogenic reservoir into which the perforated grid and sample may beimmersed for vitrification of the sample. In an embodiment, thereservoir includes an inner chamber for accommodating a first cryogenicfluid and into which the grid and sample may be immersed, and an outerchamber situated about the first chamber for accommodating a secondcryogenic fluid. The system further includes a stage, provided with atemperature substantially similar to the cryogenic reservoir, and ontowhich the grid and sample may be situated for subsequent imaging. Thesystem may also be provided with a high contrast imager, such as anelectron microscope, designed to receive the stage with the grid forimaging a relatively thin film region of the sample to permitidentification of ultrastructural biomarkers therein.

The present invention also provides a method for generating an image ofultrastructural biomarkers from a biological sample. The methodincludes, in one embodiment, providing a substantially thin film of asample to be imaged. The thin film may be generated from blotting oralternatively from sandwiching the sample between two plates. Next, thesample may be immersed in a cryogenic fluid so as to cause the sample tovitrify. This rapid vitrification allows the objects present in thesample to substantially maintain their original morphology. Oncevitrified, the sample may be transferred onto a stage for placement in ahigh contrast imager, such as a transmission electron microscope, underpositive dry pressure to minimize the risks of contamination of thesample. The transfer to the high contrast imager also includes keepingthe sample at a temperature range of from about −170° C. to about −150°C. in the imager to maintain the integrity of the sample. Thereafter, animage of the thin film sample may be generated for subsequentidentification of ultrastructural biomarkers. The generation of theimage, in one embodiment, includes producing a substantiallyartifact-free image in the absence of contrasting agents.

The method for generating an image of ultrastructural biomarkers from abiological sample may be used subsequently for screening and monitoringdiseases or disease susceptibility, evaluating drug or therapeuticefficacy, and assessing risks associated with a drug or therapeuticcandidate, among other things. In one embodiment, a vitrified biologicalsample from a test subject may initially be provided. Next, an imagefrom the vitrified sample may be generated, in a high contrast imager,for subsequent identification of ultrastructural biomarkers. Thereafter,the biomarkers from the vitrified sample may be compared to thosebiomarkers from a healthy subject or control population, for structuralor morphological variations Subsequently, the presence of structural ormorphological variations may be analyze and used as determinants orpredictors for a disease, for evaluating drug or therapeutic efficacy,or assessing risks associated with a drug or therapeutic candidate.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-C illustrate a system for use in preparing samples forsubsequent imaging and identification of ultrastructural biomarkers.

FIG. 2 illustrates a cryotransfer station onto which a sample may betransferred for subsequent placement into a high contrast imaging devicefor in imaging and identifying ultrastructural biomarkers.

FIG. 3 illustrates schematically another method for preparing samplesfor subsequent imaging and identification of ultrastructural biomarkers,in accordance with an embodiment of the present invention.

FIGS. 4A-D illustrate cryogenic TEM images of a human blood serumsample.

FIGS. 5A-B illustrate a change comparison in morphology and aggregatestate between a control sample and a diseased sample.

DESCRIPTION OF SPECIFIC EMBODIMENTS

The present invention provides, in one embodiment, a method foridentifying ultrastructural biomarkers through the use of cryogenictransmission electron microscopy (cryo-TEM), or modified freeze fracturedirect imaging (M-FFDI). In particular, the combination of cryogenicvitrification of biological samples and subsequent high contrast imagingof the samples, can preferably generate substantially artifact-freeimages, unlike those images obtained from the use conventional TEMalone. As such, the ability to visualize ‘soft’ structures that range,for instance, from between about 5 nanometers (nm) and about 500 nm,makes these artifact-free imaging techniques ideally suited for highresolution imaging of biomolecular aggregates, such as proteins, virusesand cellular organelles in their native hydrated states forultrastructural analysis. Moreover, the data obtained by cryo-TEM orM-FFDI can complement atomic level information from, for instance, X-raydiffraction (where crystals of the sample have to be produced beforeidentification—these crystals do not represent the true hydratedconfiguration in solution) and NMR, as well as micron level informationfrom light microscopy for biomarker identification.

Cryo-TEM Technique:

1. The Controlled Environmental Vitrification System

Referring now to FIGS. 1A-C, there is illustrated a ControlledEnvironmental Vitrification System (CEVS)10, such as that available fromThe Department of Chemical Engineering & Material Sciences at theUniversity of Minnesota, for use in the preparation of samples forsubsequent ultrastructural biomarker identification. In general, theCEVS 10 includes a housing H equipped with temperature and humiditycontrol. The CEVS 10 also includes a vial 11, within which a volume ofsample may be stored. The sample within the vial 11, in one embodiment,may be thermally equilibrated relative to the interior of housing H. Toextract an amount of the sample from the vial 11, a pipette 12 may beprovided adjacent to the vial 11. The CEVS 10 may also include a grid 13onto which the extracted sample may be deposited from the pipette 12.The grid 13, in one embodiment, may be a perforated disc with holes 131that are sufficiently spaced to support the deposited sample. In anembodiment, the grid 13 may be a specially prepared holey carbon gridthat is approximately 3 millimeters (mm) in diameter, if circular inshape. Of course, the grid may be designed with other geographic shapesif necessary. Such a holey carbon grid is well known in the art andincludes, among other things, a perforated copper disc having a carbonlayer, approximately 200 nm in thickness, extending across holes 131.The carbon layer, in an embodiment, is provided to decrease the diameterof the holes 131 on the grid 13, and does not completely cover theseholes 131. In accordance with an embodiment, the holes 131 may beprovided with a diameter ranging from about 1 micrometer to about 10micrometers. In this manner, when the sample is deposited onto the grid13, a thin film of the sample may be generated across the reduced-sizeholes 131. To facilitate the generation of the thin film, the CEVS 10may be provided with a blotter 14 adjacent the grid 13 to blot excessamount of the deposited sample from the surface of the grid 13, suchthat an amount sufficient for generating the thin film remains on thegrid 13. In addition, to maintain the grid 13 in position during thedeposition process and blotting process, a plunging mechanism 15 may beprovided to which the grid 13 may be affixed.

The CEVS 10 may further include a portal 16 through which the plungingmechanism 15 may extend, so as to push the grid 13 from within the CVES10, as illustrated in FIG. 1B. In this manner the grid 13 may beimmersed within a cryogenic reservoir 17 situated in proximity to theportal 16. The reservoir 17, in one embodiment, includes an innerchamber 171 within which a volume of a first cryogenic fluid may beaccommodated and into which the grid 13 along with the sample may beimmersed. In one embodiment, the first cryogenic fluid may be liquidethane, or similarly cold cryogenic fluid, such as liquid propane. Forethane, the normal melting point is about −183° C., while the normalboiling point is about −89° C. The reservoir 17 may also include anouter chamber 172 situated about the inner chamber 171 for accommodatinga volume of a second cryogenic fluid, such as liquid nitrogen, or asimilarly cold cryogenic fluid. By positioning outer chamber 172 aboutinner chamber 171, the volume of liquid ethane in inner chamber 171 maybe kept sufficiently cold and substantially close to its melting pointby the presence of the liquid nitrogen in the outer chamber 172 tomaximize heat transfer away from the sample and to allow the sample tovitrify rather than crystallize. The reservoir 17 may further include agrid holder 18 submerged within the liquid nitrogen in the outer chamber172 for subsequent placement of the grid 13 thereon.

2. Preparation of the Sample

Still looking at FIGS. 1A-C, approximately 1-10 microliters of theliquid sample may be withdrawn from vial 11 using pipette 12, andsubsequently deposited onto perforated grid 13. In one embodiment, thesample may be about 2-5 microliters in volume, and preferably, about 3microliters in volume. The sample may thereafter be blotted by blotter14, so as to leave on grid 13 a thin film having a thickness rangingfrom about 50 nm to about 200 nm spanning the holes 131. It should benoted that thickness of the thin film may not be substantially uniformthroughout. To that end, areas of the film that is relatively thin canprovide an ideal location for imaging.

The sample on grid 13 may then be immersed by plunging mechanism 15 intoreservoir 17, as shown in FIG. 1B, and in particular, into chamber 171of liquid ethane having a temperature range of from about −170° C. toabout −150° C. As noted above, the liquid ethane may be kept close toits melting point by the liquid nitrogen in the outer chamber 172 tomaximize heat transfer from the grid. Contact of the sample on the grid13 with the liquid ethane in chamber 171 can induce rapid vitrificationof the sample on grid 13 within a few milliseconds. It should beappreciated that during vitrification, liquid, such as water, in thesolution solidifies without crystallization. In this manner,substantially all of the microstructures in the sample may be preservedin their original state. The grid 13 may next be transferred from theinner chamber 171 of the reservoir 17 to the outer chamber 172 andplaced onto the grid holder 18 in liquid nitrogen.

Referring now to FIG. 2, the grid 13 may thereafter be transferred fromthe holder 18 onto a cold stage 20 in the liquid nitrogen environment ofthe outer chamber 172 of reservoir 17. Transferring the grid 13 in aliquid nitrogen environment can help to maintain the integrity of thesample. As illustrated, cold stage 20 may include a container 21 withinwhich a volume of, for instance, liquid nitrogen or any other similarlycold substance may be stored, and an arm 22 extending from the container21. Arm 22, in an embodiment, may be designed for placement within theliquid nitrogen environment of the outer chamber 172, and may be tubularin shape. As such, arm 22 may be made from a material that can withstandimmersion in liquid nitrogen. Arm 22 may also include a channel 23, soas to permit liquid nitrogen from container 21 to advance to tip 24 andmaintain the temperature of the arm 22 thereat from about −170° C. toabout −160° C., substantially well below the amorphous to crystallinephase transition temperature of about −155° C. in ice, to minimize anycompromise to the integrity of the sample. As shown in FIG. 2, arm 22may include a depression 25 towards tip 24 to provide an area onto whichgrid 13 may be placed for subsequent imaging. Cold stage 20, in oneembodiment, can be a commercially available cold stage, such as theCryotransfer System—CT3500J from Oxford Instruments.

Once the grid 13 has been transferred onto arm 22 of cold stage 20, thecold stage 20 may be inserted into, for instance, a high contrast imager(not shown), such as a TEM, under positive dry pressure to minimize therisks of contamination of the sample by, for example, atmosphericcontaminants, including moisture. The positive dry pressure may begenerated from any gas, such as nitrogen or oxygen. During imaging, suchas phase contrast imaging, the tip 24 of arm 22 continues to bemaintained at a temperature range well below the amorphous tocrystalline phase transition temperature of about −155° C. in ice, inthe electron microscope to maintain the integrity of the sample beingimaged.

M-FFDI Technique:

In samples having a viscosity that may be relatively high, i.e., greaterthan about 100 centipoise, for blotting to effectively thin down thesamples, the use of cryo-TEM may not be sufficient. As such, the presentinvention contemplates the use of M-FFDI.

Looking now at FIG. 3, in one embodiment, approximately 100 nanoliters(nL) of the sample 30 may placed onto a plate or planchette 31.Planchette 31, in an embodiment, may be a copper planchette, or may bemade from a similar material of approximately 3 mm×3 mm in size. Next, agrid 32, either standard electron microscope grid or a holey carbongrid, such as that described above, may be placed onto the planchette 31over the sample 30, so that the sample 30 may permeate across theperforations of the grid 32. In an alternate embodiment, the sample 30may initially be placed on the grid 32 and the grid 32 subsequentlyplaced onto the planchette 31. The planchettes 31, in accordance with anembodiment, may be relatively larger in size than the grid 32, so as toaccommodate the grid 32 thereon.

Thereafter, a second planchette 33 may be gently lowered onto the grid32 to sandwich the grid 32 between the two planchettes 31 and 33. Itshould be appreciated that gentle placement of the second planchette 33onto the grid 32 allows the sample 30 to be squeezed between theplanchettes 31 and 33, and spread out over the surface of the grid 32into previously unoccupied areas. Moreover, the spreading of the sample30 across of the grid 32 into previously unoccupied areas generatescertain thin film portions that can be substantially thinner inthickness than others across the perforations of the grid 32. Thepresence of the relatively thin portions can facilitate imaging of thestructures within these thin portions of the sample 30.

The planchette-grid-planchette sandwich may then be immersed into, forinstance, liquid ethane that is maintained near its melting point with atemperature range of from about −170° C. to about −160° C. Oncevitrification of the sample 30 has taken place, the copper planchettes31 and 33 may be separated (e.g., peeled apart) while they remainimmersed in the liquid ethane to remove the grid 32 therebetween. In anembodiment, a cryogenically cooled forceps (not shown) may be used toseparate the grid 32 from the two planchettes. Next, the grid 32 may bewithdrawn and stored under liquid nitrogen, for instance, on a gridholder, such as that shown in FIG. 1B, until it is ready to betransferred to cold stage 34 for direct imaging of the ultrastructuralbiomarkers within the sample 30.

This technique can be suited for preparation of highly viscous samplesand gels, where blotting may not be feasible, for subsequent highresolution imaging. In other words, those samples that cannot beprepared for imaging using cryo-TEM can be prepared using this M-FFDItechnique. This technique can also be employed to prepare samples thathave a predominant organic phase that tend to dissolve if exposeddirectly to ethane. Accordingly, by providing these two approaches forimaging, a substantially complete range of solutions or biologicalfluids that can be imaged.

The combination of cryogenic vitrification for sample preparation andthe high contrast microscopy for imaging of the sample can producereliable, substantially artifact-free direct images of ultrastructuralbiomarkers, for instance, nanoscale aggregates in solution, or of softtissue sections in their native states. It should be appreciated thatneither cryo-TEM nor M-FFDI requires the use heavy metal salts to createcontrast, thus avoiding salt-related phase transitions. In addition, thestructural information obtained from cryo-TEM or M-FFDI, when appliedto, for instance, computer based reconstruction of images obtained atdifferent angles or stage tilts, can provide three dimensional (3-D)structural information on macromolecular assemblies. Such 3-Dreconstruction is well known in the art, for example, 3-D images createdfrom CAT scans.

Although the use of cryogenic vitrification is described above inconnection with cryo-TEM and M-FFDI, it should be appreciated that suchcan be employed with other imaging approaches. For instance, cryogenicvitrification may be used in connection with Cryotoming to image andexamine ultrastructural biomarkers in various tissue samples. As anexample, a sample of about 1 mm square may be vitrified by high pressurefreezing. The vitrified sample may then be positioned and secured on,for instance, a cold aluminum pin. Thereafter, a section ofapproximately 50 nm-100 nm may be microtomed (i.e., sliced) using a colddiamond knife. This sample may subsequently be placed on a carbon-coatedelectron microscope grid, and imaged at from about −170° C. to about−150° C. on a cold stage.

EXAMPLES

In an experiment, a serum sample from a test subject was prepared usingcryogenic vitrification in the manner set forth above and subsequentlyimaged through the use of cryo-TEM. The images of the serum sample areillustrated in FIG. 4. In particular, the images are taken fromdifferent regions of the same holey carbon grid (i.e. differentrelatively thin regions of the thin film on the grid). As can be seen,the images show a relatively rich range of structures, including (a)multilamellar vesicles, (b) single and (d) compound discs, and (c)compound vesicles, all at nanoscale resolution of approximately 500 nmor less.

It should be noted that serum typically contains macromolecules, such asmetabolites, lipids, hormones, peptides, and proteins. Certain of thesebiological macromolecules can also organize into 3-D complexes, whichcan be biochemically homogeneous or heterogenous in nature. Forexamples, serum can contain many glycoproteins, glycopeptides,lipoproteins, and hormones and metabolites complexed with proteins, andlipids.

In FIGS. 5A-B, there is illustrated a comparison of the structural andphysical changes between a control sample (FIG. 5A) and a diseasedsample (FIG. 5B). Images of both can, of course, be obtained using theprotocol set forth above. It is noted that protein structures 51 in thecontrol sample can become more aggregated in the diseased sample,whereas vesicles 52 in the control sample can change shape, becomingmore elliptical or larger in size. These structural or morphologicalchanges can act as determinants, among other things, screening andmonitoring diseases.

Other Applications

In accordance with an embodiment of the present invention,ultrastructural biomarkers identified through the use of cryo-TEM orM-FFDI may be used for a wide variety of applications, for example, tomake early disease screening (i.e., prediction, susceptibility), diseasemonitoring, early markers of drug related toxicity, and drug efficacy,among others. Other applications that can be imagined include those fordiagnostic, therapeutic, prophylactic, drug discovery, and patientstratification purposes.

A biomarker or biological marker is defined by the FDA as acharacteristic that is objectively measured and evaluated as anindicator of normal biologic or pathogenic processes or pharmacologicalresponses to a therapeutic intervention.

In diseased individuals, the composition of, for instance, serumcomponents can be altered due to cell proliferation, metabolic,hormonal, inflammatory or secretory changes, thus impacting thestructure and morphology of the resulting imaged components. As thereare numerous diseases where ultrastructural abnormalities occur in cellorganelles, tissue structures, and biological fluids, the utilization ofultrastructural analysis of components therein can reveal criticalbiomarkers associated with these diseases. Moreover, by screening andcomparing biomarkers from a sample of a test subject to those biomarkersfrom a healthy subject or control population for structural ormorphological variations, the presence of variations in theultrastructural biomarkers from the test subject sample, in oneembodiment, can act as determinants or predictors of disease, diseasepredisposition, and disease susceptibility.

Examples of biological fluids from which ultrastructural abnormalitiescan be observed include, but are not limited to blood, mucosa, plasma,serum, cerebral spinal fluid, spinal fluid, joint fluid, urine, saliva,bile, pancreatic fluid, peritoneal fluid, lung fluid, alveolar sacfluid, sinus fluid, lachrymal fluid, nasal mucous and fluid,intrathoracic fluid, gastric fluid, gastrointestinal fluid, ovarianfluid, testicular, prostrate fluid, uterine fluid, cystic fluid, renalfluid, brain fluid, opthalmic fluid, tear, ear fluid, auditory canalfluid, subcutaneous or muscular fluid.

Examples of cell organelles and tissue structures within whichultrastructural abnormalities can occur include plasma membrane,organelle membranes, basement membrane, extracellular matrix,intercellular organelles, intercellular structures, intracellularmembranes, intracellular organelles, cell-cell junctions, cell-celladhesion, gap junctions, tight junctions, nucleus, nucleolus, nuclearmembrane, nuclear pore, chromosomes, chromatin, ribosomes,polyribosomes, monosomes, cellular proteins, cellular protein complexes,cellular protein subunits, extracellular proteins, extracellular proteincomplexes, extracellular protein subunits, secretory proteins, secretedprotein complexes, secretory protein subunits, secreted intracellular orextracellular protein aggregates, golgi, lysosomes, mitochondria,endosomes, mitochondrial membranes, peroxisomes, endoplasmic reticulum,mRNA, DNA, tRNA, rRNA, small RNA, proteosomes, vacuoles, intracellularand extracellular vesicles, cavity, and droplets, cellular lipids orcarbohydrates, cellular lipid or carbohydrate complexes, cellularlipoproteins, cellular glycoproteins, intracellular and extracellularlipids, extracellular lipoprotein complexes, extracellular lipoproteinsubunits, secreted proteins, secreted protein subunits, lipoprotein orglycoprotein aggregates can reveal critical biomarkers associated withthese diseases.

Moreover, it is well established that structural and morphologicalvariations in secreted components of biological fluids, such as serum,precede or occur simultaneously with functional changes. Accordingly,the ability to monitor the structural or morphological changes inaggregates present in biological fluids in their native, hydrated statesat nanoscale resolution, and which can be correlated to functional andphenotypic changes has the potential for early and simple detection ofdisease, classification of disease sub-categories, and monitoring ofdisease progression. In one embodiment, to effectively monitor theprogress of a disease via an image-based platform, such as that employedin the present invention, an accurate, precise and temporally contiguouspicture of the progress of the disease is needed. The method of thepresent invention can provide an accurate and precise image of theultrastructural biomarkers from samples taken from a subject over aperiod of time. As a result, these images may be compared against oneanother for any structural or morphological changes in the biomarkersbeing observed to determine and monitor the progression of the disease.

The ultrastructural biomarkers identified can also be employed for drugor biological therapeutics screening. For example, in cell-based or invitro drug screening, any intracellular or extracellular markers ofchange can be detected and utilized as a marker of drug or therapeuticefficacy or an indicator that the drug target is being hit. Inparticular, in one embodiment, different drugs, candidate drugs ortherapeutics may be administered to test subjects, and the side effects,including desired effects, toxicity, adverse effects or serious adverseeffects, may be documented. Any conventional metrics of side effectseverity can be used. In addition, before and after drug administration,the biomarkers may be identified and analyzed to determine which of thebiomarkers has changed. In this way, the biomarkers affected by eachdrug can be correlated with the particular desirable and undesirableeffects of the drug.

It is anticipated that new drugs being developed will have fewer adverseeffects due to extensive use of biomarkers to identify adverse events inpreclinical animal models or in clinical trial patients. As additionalgenerations of drugs continues to be developed, the list of relevantbiomarkers and their changes can be refined further. In addition, as itbecomes clear whether each biomarker is indicative of desired orundesired effects, more information about the mechanisms of drug actionare learned, helping to direct development of next generation drugs.Accordingly, these ultrastructural biomarkers can allow for themonitoring and evaluating of drug or therapeutic safety and efficacyduring discovery, preclinical and all levels of clinical trials, as wellas post sales monitoring and testing. Furthermore, a similar approachmay be used to determine and evaluate patient response or response rate,as well as clinical trial participant response or response rate.

Similarly, the above protocol can be employed to generate informationthat can lead to the understanding of the risks of adverse events,toxicity or serious adverse events associated with marketed drugs ortherapeutics, drug or therapeutic candidates, as well as risks for drugattrition. Such an understanding can assist in a decision making processduring clinical development, thereby driving informed stop/go decisionsearly in, or prior to clinical development. The information may also beused, in an embodiment, in designing and developing drugs ortherapeutics that can be tailored to address only relevant diseasemechanisms while causing fewer adverse effects.

The present invention, in addition to being able to resolveultra-structural features in the morphology of cells, cellularorganelles, and extracellular matrix, can also employ Cryo-TEM andM-FFDI to detect macromolecular structural differences in lipiddroplets, vesicles, and other structural components in biologicalfluids.

Furthermore, the present invention permits, in an embodiment,identification of the spatial positions of proteins in a larger assemblyor changes of protein complex morphology. This is important becauseproper assembly can be critical to the functioning of the proteincomplexes and cell organelles. In particular, there are potentialchanges in morphology and aggregates in different stages of diseasewhich can change size or size distribution. Since these changes arephysical, the identification process employed by a method of the presentinvention does not require any a priori knowledge of specific biologicaltargets. Accordingly, sole reliance on biochemical assays can beeliminated.

The high resolution images of these ultrastructures, ranging fromnanometers to micrometers in size, thus provide clear indications thatcryogenic vitrification and high contrast imaging achieved throughcryo-TEM or M-FFDI can generate a powerful tool for analyzing thesenanostructures and changes to these nanostructures in biologicalsamples. Whether, the sample is fluid or viscous, the provision of theeither cryo-TEM or M-FFDI, as disclosed herein, can create broadcapability to examine relevant bio-samples under conditions that mostclosely resemble their native states. For instance, biomarkers that arefrom any of parts of the human body, including any viruses, bacteria orother pathogens residing in any part of the human body can be identifiedemploying the methods of the present invention.

Moreover, since the present invention involves utilization ofresolutions relatively far beyond those traditionally used, thepotential for discovery of early changes of structural markers can besubstantially high. Thus, the use of cryo-TEM and M-FFDI coupled withimage analysis can provide a novel and high resolution approach for theidentification of ultrastructural biomarkers. Furthermore, such anapproach has the potential to change the paradigm and dramaticallyreduce the cost associated with biomarker discovery and validation, byproviding a robust and relatively sensitive approach to diagnosing andmonitoring diseases, while simultaneously reducing drug developmentcosts.

Although the above description has been provided in the context of humansubjects, it can be equally well applied to animal models, particularlythose with immune systems similar to the human immune systems. Forinstance, suitable animals include mice, rats, and rabbits.

While the invention has been described in connection with the specificembodiments thereof, it will be understood that it is capable of furthermodification. Furthermore, this application is intended to cover anyvariations, uses, or adaptations of the invention, including suchdepartures from the present disclosure as come within known or customarypractice in the art to which the invention pertains, and as fall withinthe scope of the appended claims.

1. A system for identifying ultrastructural biomarkers from a biologicalsample, the system comprising: a grid onto which a biological sample tobe imaged may be placed; a cryogenic reservoir into which the perforatedgrid and sample may be immersed for vitrification of the sample; astage, provided with a temperature substantially similar to thecryogenic reservoir, and onto which the grid and sample may be situatedfor subsequent imaging; and a high contrast imager designed to receivethe stage with the grid for imaging a region of the thin sample forsubsequent identification of ultrastructural biomarkers.
 2. A system asset forth in claim 1, wherein the grid includes a plurality of holesacross which the sample may extend, so as to enhance generation of athin film thereacross.
 3. A system as set forth in claim 1, wherein thegrid further includes a first plate onto which the grid may bepositioned and a second plate for placement onto the grid.
 4. A systemas set forth in claim 3, wherein the first and second plates act tospread the sample across the grid, so as to generate certain portionsthat can be substantially thinner in thickness than others across thegrid.
 5. A system as set forth in claim 3, further includingcryogenically cooled forceps to permit separation of the grid from thefirst and second plates.
 6. A system as set forth in claim 1, whereinthe cryogenic reservoir includes an inner chamber for accommodating afirst cryogenic fluid, and an outer chamber situated about the innerchamber for accommodating a second cryogenic fluid.
 7. A system as setforth in claim 6, wherein the first and second cryogenic fluids aredifferent and the presence of the second cryogenic fluid in the outerchamber helps to maintain the first cryogenic fluid substantially closeto its melting point.
 8. A system as set forth in claim 6, wherein thecryogenic reservoir further includes a grid holder positioned within theouter chamber for placement of the grid thereon prior to transferenceonto the stage.
 9. A system as set forth in claim 1, wherein the stageincludes a container for accommodating a cryogenic fluid, and an armextending from the container for placement of the grid thereon, the armhaving a channel along which cryogenic fluid from the container may flowtoward the grid, so as to maintain the temperature of the gridsubstantially similar to that of the cryogenic reservoir.
 10. A systemas set forth in claim 1, wherein the high contrast imager can generatesubstantially artifact-free images in the absence of contrasting agents.11. A system as set forth in claim 1, further including a positivepressure environment within which the grid may be transferred onto thestage and into the high contrast imager, so as to maintain the integrityof the sample and to minimize risks of contamination of the sample. 12.A system as set forth in claim 1, wherein the biomarkers includecomponents from one of intracellular organelles or components,extracellular organelles or components, tissue components, andbiological fluids.
 13. A system as set forth in claim 12, wherein theidentified ultrastructural organelles or components, extracellularorganelles or components can be used to evaluate, determine, or predictdrug or therapeutic efficacy, patient response or response rate, orclinical trial participant response or response rate.
 14. A system asset forth in claim 12, wherein the identified ultrastructuralbiomarkers, when compared to ultrastructural biomarkers from healthy orcontrol intracellular organelles or components, extracellular organellesor components, tissue components, and biological fluid can act to assessrisks for adverse events, toxicity, or serious adverse events associatedwith drug or therapeutic candidates in preclinical development, inanimal models, or in clinical development, as well as risks for drugattrition in preclinical development, animal models, clinicaldevelopment or in marketed drugs. 15-41. (canceled)